Enniatin and Beauvericin Biosynthesis in Fusarium Species: Production Profiles and Structural Determinant Prediction.
ABSTRACT: Members of the fungal genus Fusarium can produce numerous secondary metabolites, including the nonribosomal mycotoxins beauvericin (BEA) and enniatins (ENNs). Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) that contains both peptide synthetase and S-adenosyl-l-methionine-dependent N-methyltransferase activities. Several Fusarium species can produce ENNs, BEA or both, but the mechanism(s) enabling these differential metabolic profiles is unknown. In this study, we analyzed the primary structure of ESYN1 by sequencing esyn1 transcripts from different Fusarium species. We measured ENNs and BEA production by ultra-performance liquid chromatography coupled with photodiode array and Acquity QDa mass detector (UPLC-PDA-QDa) analyses. We predicted protein structures, compared the predictions by multivariate analysis methods and found a striking correlation between BEA/ENN-producing profiles and ESYN1 three-dimensional structures. Structural differences in the ? strand's Asn789-Ala793 and His797-Asp802 portions of the amino acid adenylation domain can be used to distinguish BEA/ENN-producing Fusarium isolates from those that produce only ENN.
Project description:Fungi from the Hypocreales order synthesize a range of toxic non-ribosomal cyclic peptides with antimicrobial, insecticidal and cytotoxic activities. Entomopathogenic Beauveria, Isaria and Cordyceps as well as phytopathogenic Fusarium spp. are known producers of beauvericins (BEAs), beauvenniatins (BEAEs) or enniatins (ENNs). The compounds are synthesized by beauvericin/enniatin synthase (BEAS/ESYN1), which shows significant sequence divergence among Hypocreales members. We investigated ENN, BEA and BEAE production among entomopathogenic (Beauveria, Cordyceps, Isaria) and phytopathogenic (Fusarium) fungi; BEA and ENNs were quantified using an LC-MS/MS method. Phylogenetic analysis of partial sequences of putative BEAS/ESYN1 amplicons was also made. Nineteen fungal strains were identified based on sequence analysis of amplified ITS and tef-1? regions. BEA was produced by all investigated fungi, with F. proliferatum and F. concentricum being the most efficient producers. ENNs were synthesized mostly by F. acuminatum, F. avenaceum and C. confragosa. The phylogeny reconstruction suggests that ancestral BEA biosynthesis independently diverged into biosynthesis of other compounds. The divergent positioning of three Fusarium isolates raises the possibility of parallel acquisition of cyclic depsipeptide synthases in ancient complexes within Fusarium genus. Different fungi have independently evolved NRPS genes involved in depsipeptide biosynthesis, with functional adaptation towards biosynthesis of overlapping yet diversified metabolite profiles.
Project description:Beauvericin (BEA) and enniatins (ENNs) are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1? gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.
Project description:In 2017-2018, extensive symptoms of sudden decline and fruit rot were observed on date palms in southern Tunisia. Samples of diseased plants were randomly collected in six localities. Based on morphological identification, <i>Fusarium</i> was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative <i>Fusarium</i> strains at species level and investigate their phylogenetic relationships. The main species detected was <i>Fusarium</i> <i>proliferatum</i>, and at a much lesser extent, <i>Fusarium</i> <i>brachygibbosum</i>, <i>Fusarium</i> <i>caatingaense</i>, <i>Fusarium</i> <i>clavum</i>, <i>Fusarium</i> <i>incarnatum</i><i>,</i> and <i>Fusarium</i> <i>solani</i>. Pathogenicity on the <i>Deglet</i> <i>Nour</i> variety plantlets and the capability to produce mycotoxins were also assessed. All <i>Fusarium</i> species were pathogenic complying Koch's postulates. <i>Fusarium</i> <i>proliferatum</i> strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All <i>F.</i> <i>brachygibbosum</i> strains produced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. <i>Fusarium</i> <i>caatingaense</i>, <i>F.</i> <i>clavum</i>, <i>F.</i> <i>incarnatum</i> produced only BEA. <i>Fusarium</i> <i>solani</i> strains produced MON, BEA, and ENNs. This work reports for the first time a comprehensive multidisciplinary study of <i>Fusarium</i> species on date palms, concerning both phytopathological and food safety issues.
Project description:Recent surveys report the occurrence of <i>Aspergillus</i> and <i>Penicillium</i> metabolites (aflatoxins (AFLs), ochratoxin A (OTA), cyclopiazonic and mycophenolic acids (MPA), sterigmatocystin (STC), citrinin), <i>Fusarium</i> (trichothecenes, zearalenone (ZEA), fumonisins (FBs), enniatins (ENNs)) and <i>Alternaria</i> (alternariol (AOH), its methyl ether (AME), tentoxin (TE), and tenuazonic acid (TNZ)) toxins in dry <i>Camellia sinensis</i> and herbal tea samples. Since tea is consumed in the form of infusion, correct risk assessment needs evaluation of mycotoxins' transfer rates. We have studied the transfer of AFLs, OTA, STC, deoxynivalenol (DON), ZEA, FBs, T-2, and HT-2 toxins, AOH, AME, TE, ENN A and B, beauvericin (BEA), and MPA from the spiked green tea matrix into an infusion under variation of preparation time and water characteristics (total dissolved solids (TDS) and pH). Analytes were detected by HPLC-MS/MS. The main factors affecting transfer rate proved to be mycotoxins' polarity, pH of the resulting infusion (for OTA, FB2, and MPA) and matrix-infusion contact period. The concentration of mycotoxins increased by 20-50% within the first ten minutes of infusing, after that kinetic curve changed slowly. The concentration of DON and FB2 increased by about 10%, for ZEA, MPA, and STC it stayed constant, while for T-2, TE, AOH, and AFLs G1 and G2 it went down. Maximum transfer correlated well with analytes polarity. Maximum transfer of ENNs, BEA, STC, ZEA, and AOH into infusion was below 25%; AFLs-25-45%; DON, TE, and T-2 toxins 60-90%, FB1-80-100%. The concentration of OTA, MPA, and FB2 in the infusion depended on its pH. At pH about four, 20%, 40%, and 60% of these toxins transferred into an infusion, at pH about seven, their concentrations doubled. Water TDS did not affect transfer significantly.
Project description:In this study, the occurrence of multiple fungal metabolites including mycotoxins was determined in four different winter wheat varieties in a field experiment in Croatia. One group was naturally infected, while the second group was inoculated with a Fusarium graminearum and F. culmorum mixture to simulate a worst-case infection scenario. Data on the multiple fungal metabolites including mycotoxins were acquired with liquid chromatography with mass spectrometry (LC-MS/MS) multi-(myco)toxin method. In total, 36 different fungal metabolites were quantified in this study: the Fusarium mycotoxins deoxynivalenol (DON), DON-3-glucoside (D3G), 3-acetyldeoxynivalenol (3-ADON), culmorin (CULM), 15-hydroxyculmorin, 5-hydroxyculmorin, aurofusarin, rubrofusarin, enniatin (Enn) A, Enn A1, Enn B, Enn B1, Enn B2, Enn B3, fumonisin B1, fumonisin B2, chrysogin, zearalenone (ZEN), moniliformin (MON), nivalenol (NIV), siccanol, equisetin, beauvericin (BEA), and antibiotic Y; the Alternaria mycotoxins alternariol, alternariolmethylether, altersetin, infectopyron, tentoxin, tenuazonic acid; the Aspergillus mycotoxin kojic acid; unspecific metabolites butenolid, brevianamid F, cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and tryptophol. The most abundant mycotoxins in the inoculated and naturally contaminated samples, respectively, were found to occur at the following average concentrations: DON (19,122/1504 µg/kg), CULM (6109/1010 µg/kg), 15-hydroxyculmorin (56,022/1301 µg/kg), 5-hydroxyculmorin (21,219/863 µg/kg), aurofusarin (43,496/1266 µg/kg). Compared to naturally-infected samples, Fusarium inoculations at the flowering stage increased the concentrations of all Fusarium mycotoxins, except enniatins and siccanol in Ficko, the Aspergillus metabolite kojic acid, the Alternaria mycotoxin altersetin, and unspecific metabolites brevianamid F, butenolid, cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In contrast to these findings, because of possible antagonistic actions, Fusarium inoculation decreased the concentrations of the Alternaria toxins alternariol, alternariolmethylether, infectopyron, tentoxin, tenuazonic acid, as well as the concentration of the nonspecific metabolite tryptophol.
Project description:Pineapple (Ananas comosus var. comosus) is an important perennial crop in tropical and subtropical areas. It may be infected by various Fusarium species, contaminating the plant material with mycotoxins. The aim of this study was to evaluate Fusarium species variability among the genotypes isolated from pineapple fruits displaying fungal infection symptoms and to evaluate their mycotoxigenic abilities. Forty-four isolates of ten Fusarium species were obtained from pineapple fruit samples: F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incarnatum, F. oxysporum, F. polyphialidicum, F. proliferatum, F. temperatum and F. verticillioides. Fumonisins B1-B3, beauvericin (BEA) and moniliformin (MON) contents were quantified by high-performance liquid chromatography (HPLC) in pineapple fruit tissue. Fumonisins are likely the most dangerous metabolites present in fruit samples (the maximum FB1 content was 250 μg g(-1) in pineapple skin and 20 μg ml(-1) in juice fraction). In both fractions, BEA and MON were of minor significance. FUM1 and FUM8 genes were identified in F. fujikuroi, F. proliferatum, F. temperatum and F. verticillioides. Cyclic peptide synthase gene (esyn1 homologue) from the BEA biosynthetic pathway was identified in 40 isolates of eight species. Based on the gene-specific polymerase chain reaction (PCR) assays, none of the isolates tested were found to be able to produce trichothecenes or zearalenone.
Project description:Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate food and feed resources, and are capable of inducing a wide range of toxicity. Here, we studied the developmental and behavioral toxicity in zebrafish (Danio rerio) embryos and larvae exposed to three mycotoxins: beauvericin (BEA), Enniatin A (ENN A), and Ennitain B (ENN B). Zebrafish embryos were collected after fertilization, treated individually from 1 to 6 dpf with BEA at 8, 16, 32 and, 64 μM and for both enniatins at 3.12, 6.25, 12.5 and, 25 μM. Mixture of mycotoxins were assayed as follows: i) for BEA + ENN A and BEA + ENN B at [32 + 12.5] μM and [16 + 6.25] μM; ii) for ENN A + ENN B at [12.5 + 12.5] μM and [6.25 + 6.25] μM and, iii) for BEA + ENN A + ENN B at [32 + 12.5 + 12.5] μM and [16 + 6.25 + 6.25] μM. Response was collected after a white light-flash intermittent coming on for 5 s during 2 h with a imaging platform. Outcomes measured were: time to death, response to light, and circadian rhythm. This last outcome was measured in a plate where embryos had evolved in natural intervals of light and dark until day 7 or in a plate maintained in darkness. Images of all stages and evolution were collected. Results indicated that mycotoxins induced toxicity at the concentrations tested. All exposed zebrafish induced developmental defects, specifically hatching time and motion activity. After exposure, fish showed enhanced baseline activity but they lost their responsiveness to light.
Project description:Celiac disease (CD) is a genetic-based autoimmune disorder which is characterized by inflammation in the small intestinal mucosa due to the intolerance to gluten. Celiac people should consume products without gluten, which are elaborated mainly with maize or other cereals. Contamination of cereals with mycotoxins, such as fumonisins (FBs) and aflatoxins (AFs) is frequently reported worldwide. Therefore, food ingestion is the main source of mycotoxin exposure. A new analytical method was developed and validated for simultaneous analysis of 21 mycotoxins in gluten-free pasta, commonly consumed by celiac population as an alternative to conventional pasta. Ultrahigh-performance liquid chromatography coupled to quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) was used for analyte separation and detection. The mycotoxins included in this work were those widely reported to occur in cereal samples, namely, ochratoxin-A (OTA), aflatoxins (AFB1, AFB2, AFG1 and AFG2), zearalenone (ZON), deoxynivalenol (DON), 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol (3-AcDON and 15-AcDON, respectively), nivalenol (NIV), neosolaniol (NEO), fusarenone-X, (FUS-X), T-2 toxin (T-2) and HT-2 toxin (HT-2), fumonisin B1 and B2 (FB1 and FB2, respectively), enniatins (ENN A, ENN A1, ENN B and ENN B1) and beauvericin (BEA). The validated method was successfully applied to 84 gluten-free pasta samples collected from several local markets of Campania region (Italy) during September to November 2020 to monitor the occurrence of mycotoxins and to assess the exposure to these food contaminants. A significant number of samples (95%) showed mycotoxin contamination, being <i>Fusarium</i> mycotoxins (FB1, ZON and DON) the most commonly detected ones. Regarding the risk assessment, the higher exposures were obtained for NIV, DON and FB1 for children and teenagers age group which can be explained due to their lower body weight.
Project description:Totals of 158 corn and corn-based samples and 291 wheat and wheat-based samples from Shandong province, China in 2017 were analyzed for five mycotoxins including beauvericin (BEA), enniatin A (ENA), enniatin A? (ENA?), enniatin B (ENB), and enniatin B? (ENB?) by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). BEA was the predominant toxin detected, followed by ENB, ENA?, ENA, and ENB?. Corn and corn-based samples were more easily contaminated by BEA with an average concentration of 65.26 µg/kg, compared with that in wheat and wheat-based samples (average = 0.41 µg/kg). Concentrations of BEA, ENA, and ENB? in corn kernels, flours, and flakes were significantly different (Kruskal?Wallis Test, <i>p</i> < 0.05), as well as for BEA, ENA, ENB, and ENB? in wheat kernels, flours, and noodles (Kruskal?Wallis test, <i>p</i> < 0.05). Furthermore, 59.5% (94/158) and 59.8% (174/291) corn- and wheat-based samples were co-contaminated by at least two mycotoxins, respectively. Positive correlations in concentrations were observed in corn between levels of ENA and ENB?, ENA and ENB, ENA? and ENB?, as well as in wheat between BEA and ENA, BEA and ENA?, BEA and ENB, BEA and ENB?, ENA and ENA?, ENA and ENB, ENA and ENB?, ENA? and ENB, ENA? and ENB?, and ENB and ENB?. These results demonstrate that co-contamination of BEA and enniatins (ENNs) in corn- and wheat-based samples from Shandong, China is very common. More data on the contamination of five mycotoxins in cereal and cereal-based samples nationwide are needed.
Project description:Beauvericin (BEA) and deoxynivalenol are toxins produced by <i>Fusarium</i> species that can contaminate food and feed. The aim of this study was to assess the effects of these mycotoxins on the maturation of oocytes from gilts and sows. Furthermore, the antioxidant profiles in the oocytes' environment were assessed. Cumulus-oocyte-complexes (COCs) from gilts and sows were exposed to beauvericin (BEA) or deoxynivalenol (DON) and matured in vitro. As an extra control, these COCs were also exposed to reactive oxygen species (ROS). The maturation was mostly impaired when oocytes from gilts were exposed to 0.02 μmol/L DON. Oocytes from sows were able to mature even in the presence of 5 μmol/L BEA. However, the maturation rate of gilt oocytes was already impaired by 0.5 μmol/L BEA. It was observed that superoxide dismutase (SOD) and glutathione (GSH) levels in the follicular fluid (FF) of gilt oocytes was higher than that from sows. However, the expression of SOD1 and glutathione synthetase (GSS) was higher in the oocytes from sows than in those from gilts. Although DON and BEA impair cell development by diverse mechanisms, this redox imbalance may partially explain the vulnerability of gilt oocytes to these mycotoxins.